1. Field of the Invention
This invention relates to an enzyme immunoassay highly sensitive to a trace amount of human G-CSF, especially, in body fluids.
2. Description of Related Art
G-CSF is a hematopoietic factor which has been found in the culture of a human bladder cancer cell line 5637 (ATCC HT8-9) (Welt et al., Proc. Natl. Acad. Sci. U.S.A., 82, 1526-1530 (1985)). The DNA sequence of its gene has been determined (JP-A-63-50636), which has made it possible to produce the G-CSF by the recombinant DNA techniques.
It has been reported that G-CSF is a factor which acts on myeloid stem cells and enhances their differentiation into neutrophilic leukocytes (Metcalf et al., Blood, 67 (1), 37-45 (1986)), and that it also stimulates functions of differentiated neutrophilic leukocytes such as phagocytosis, O.sub.2.sup.- -production and the like (Yuo et al., Blood, 70 (2), 404-411 (1987)). Also, the G-CSF can effectively enhance the restoration of neutrophilic leukocytes when administered to patients with the clinical condition such as neutropenia which is caused by administration of chemotherapeutic drugs. When the G-CSF is applied clinically, it is necessary to clarify the correlation between a therapeutic effect and a G-CSF level in biological fluids. For the purpose, the level of G-CSF in the biological fluids must be determined accurately.
In addition, it has been reported that the G-CSF exists at either higher or lower concentrations in the sera of patients with hematological disorders in comparison with those of healthy persons (Watari et al., Blood, 73, 117-122 (1989); Ohmori et al., Biotherapy, 4, 147-153 (1992); International Journal of Hematology, vol.57, Supplement No.1, p.235). The highly sensitive measurement of G-CSF will therefore become an effective means, for instance, in the investigations on the relationship between the G-CSF level in the body and the etiology associated with G-CSF.
Interference is a general problem in the measurement of components in the body fluid such as plasma. This is a phenomenon that a body fluid component makes interference with an antigen-antibody reaction in a non-specific or specific fashion to generally cause the inhibition of the reaction, resulting in a serious decrease in a sensitivity for measurement. The patent application JP-A-2-287257 which has been laid open to the public discloses a method for the measurement of G-CSF in which a surfactant as well as sodium ethylenediaminetetraacetate is contained in an antigen-antibody reaction solution as a means to eliminate an interference in body fluid components.
The reported methods for the measurement of G-CSF in body fluids are divided into (1) bioassay, (2) extraction method, and (3) immunoassay.
In (1) bioassay, the method has been reported in which a serum sample is dialyzed against an acidic buffer solution and then a sodium chloride solution, followed by cultivation of the dialysate in admixture with NFS-60 cells having the ability of G-CSF-dependent proliferation, after which the concentration of G-CSF is determined through the measurement of a .sup.3 H-thymidine uptake into the cells (Shirafuji et al., Exp. Hematol., 17, 116-119 (1989)).
In (2) extraction method, lipids and almost proteins are removed from a plasma sample by mixing it with an organic solvent. Next, G-CSF-containing substances in the plasma sample are absorbed on an ion exchange resin in order to separate G-CSF from interfering substances by utilizing the difference in their affinities for the resin, thereby recovering G-CSF. Thereafter, to the recovered G-CSF are added a radioactively labeled G-CSF and an antibody to G-CSF, and the radioactivity of the resulting G-CSF/antibody complex is measured in order to determine the amount of G-CSF based on the measured value.
In (3) measurement of G-CSF by immunoassay, the radioimmunoassay (RIA) in which an radioisotope is used as a marker (Tanaka et al., J. Pharmaco-bindyn, 15, 359-366 (1992)) has been reported as well as the enzyme immunoassay (EIA) in which an enzyme is used (Motojima et al., J. Immunol.Methods, 118, 187-192 (1989); Ohmori et al., Res. Exp. Med., 189, 163-171 (1989); International Journal of Hematology, vol.56, Supplement No.1, p.202; The Japanese Journal of Clinical Hematology, vol.33, Supplement No.10, p.1544; Tanaka et al., J. Pharm. Exp. Ther., 255, 724-729 (1990); Kuwabara et al., J. Pharmacobio-Dyn., 15, 121-129 (1992); JP-A-5-115297) .
EIA is a method in which an antibody directed against an antigen to be measured is immobilized on a solid phase followed by reaction with the antigen, and the solid phase obtained is washed and then reacted with an enzyme-labeled antibody to determine the amount of the antigen (Ishikawa et al., Rinsho Kagaku (Clinical Chemistry), vol.3, p.374 (1974)) .
In comparison with the above methods (1) and (2), EIA is regarded as being an accurate and simple method because it is superior in specificity and because it does not require any complex pretreatments of samples to be assayed.
Examples of the antibody to be used in EIA include polyclonal antibodies obtained from anti-sera from immunized animals, and monoclonal antibodies obtained from the supernatant of the culture of hybridomas produced by fusion of mouse spleen cells with myeloma cells. With regard to EIA of G-CSF, methods in which a polyclonal or monoclonal antibody to the G-CSF is used have been reported.
To improve the sensitivity of the EIA for G-CSF, several methods have been reported: the method in which non-specific binding is repressed by use of a digested antibody fragment as a labeled antibody; and the method in which enzymatic activity is measured by a highly sensitive fluorescence technique (International Journal of Hematology, vol.56, Supplement No.1, p.202) or chemiluminescence technique (The Japanese Journal of Clinical Hematology, vol.33, Supplement No.10, p.1544).
In studying the relationship between the G-CSF level in the body and the etiology associated with G-CSF, it is important to accurately know normal or lower G-CSF levels in the body fluids from healthy persons or patients respectively. For example, it has been reported that the serum G-CSF level is distributed around 10 pg/ml in healthy persons, whilst it is about 3 pg/ml in patients with hematological disorders (International Journal of Hematology, vol.57, Supplement No.1, p.235). However, most of the assays reported can not detect a lower level of G-CSF. In only one example of EIA reported which can accurately determine a G-CSF level lower than that in healthy persons (The Japanese Journal of Clinical Hematology, vol.33, Supplement No.10, p.1544), polyclonal antibodies are used as first and second antibodies. This method, however, has the disadvantage that the number of measurements is limited because of the limitation of the availability of the antibodies having the same performance.
In the case of monoclonal antibody, since the quantity of the antibody obtained is theoretically infinite, the monoclonal antibody has the advantage that the number of measurements is not limited when applied to EIA. Especially, it is desirable to use a monoclonal antibody as a first antibody to be bound to an EIA solid phase because the larger amount of a monoclonal antibody is used in the EIA in comparison with the labeled antibody.
Although there is a report on an EIA using a monoclonal antibody which can detect the normal level of G-CSF in healthy persons (JP-A-5-115297), nothing is yet known about the similar method which can accurately measure both normal and lower levels.
On the other hand, the needed amount of a second antibody to be used in EIA is generally smaller than that of a first antibody to be immobilized on a solid phase. For example, while the amount of IgG which binds to a 96-well microplate is about 30 pmol/100 .mu.l per measurement, the amount of a second antibody used in EIA of human ferritin is 5 fmol per measurement (Hasida et al., J. Biochem., 108, 960-964 (1990)) . Also, a polyclonal antibody having a plurality of antigen-binding sites may be useful as a labeled antibody for the construction of a highly sensitive assay system, because the number of second antibody molecules to be bound per antigen molecule captured on the solid phase becomes plural.
In addition, a problem of the insufficient sensitivity which is resulted from the interference with blood components occurs often when EIA systems are to be established. This has been reported, for example, on the cytokine EIA (Ida et al., J. Immunol. Methods, 156, 27-38 (1992)). In consequence, when clinical applications of G-CSF are taken into consideration, it is necessary to develop an EIA for the highly sensitive measurement of G-CSF, in which a monoclonal antibody that is not interfered with any body fluid components can be used to accurately measure normal or lower G-CSF levels in healthy persons or patients, respectively.